Cryopreservation Techniques for Endangered Fauna Genetic Banks

Abstract

Cryopreservation of genetic material — sperm, oocytes, embryos, somatic cells, and DNA — from endangered fauna
provides an insurance policy against extinction and a resource for future assisted reproduction and genetic rescue
programmes, yet its application across taxonomic groups remains highly uneven, with mammals disproportionately
well-represented relative to amphibians, reptiles, and invertebrates. This study evaluated and compared cryopreservation
protocol efficiency across five tissue types and four taxonomic groups (mammals, birds, reptiles, amphibians) for 28
endangered species at the European Endangered Species Genome Bank (EEGB) in Zurich, Vienna, and Rome. Protocol
performance was assessed by post-thaw viability (live-dead staining), motility (CASA analysis for sperm), DNA integrity
(comet assay), and functional fertility (in vitro fertilisation rate for sperm and oocytes). Across 284 cryopreservation runs,
mean post-thaw viability was 68.4 ± 12.8% for sperm, 54.2 ± 14.4% for oocytes, 72.4 ± 8.4% for somatic cells, 84.6 ±
6.8% for blood-derived DNA, and 48.2 ± 16.4% for embryos. Mammalian tissue showed significantly higher post-thaw
viability across all tissue types than amphibian tissue (mean 78.4% vs. 42.8%; t = 8.42, p < 0.001), reflecting both
protocol optimisation bias and intrinsic biological differences in cryosensitivity. Dimethyl sulfoxide (DMSO) at 10% v/v
outperformed glycerol (15% v/v) and ethylene glycol (1.5 mol/L) as cryoprotectant for reptile and amphibian somatic cells
(post-thaw viability: 58.4 ± 8.4% vs. 38.4 ± 9.6% vs. 44.2 ± 10.8%; ANOVA F = 12.4, p < 0.001). Species-specific
protocol optimisation improved mean post-thaw viability by 18.4 ± 4.2% relative to generic mammalian protocols across
the 12 non-mammalian species tested. These results provide evidence-based cryopreservation protocol
recommendations for endangered fauna genetic banks and identify amphibian and reptile taxa as priority targets for
protocol development under the CBD Kunming-Montreal Global Biodiversity Framework Target 4.